Morphological examination

The shallow detection of cysticercosis at the slaughterhouse is the same used since more than a century without any remarkable positive changes. In the contrary, the new legislations for meat inspection concerning T. saginata cysticercosis are becom-ing more superficial and less reproducible, because there are fewer sites to be in-spected according to the new rules.

Site of the cysts: the sites of the diagnosed cysts in this thesis were concentrated in

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cysts in the slaughterhouse Berlin/Spandau (GOETZE 1989) from 1981 to 1988 were also mainly in the masseter muscle (71.1%) and the heart (27.1%) respectively. De-tectable cysts in other sites like diaphragm or sublingual muscle harboured just 1.8%.

These previous results are comparable with those found in the present study.

Nevertheless, there is clear evidence that the established meat inspection methods, i.e. the inspection of predilection sites, are imperfect for the detection of T. saginata cysticercosis. KOSMINKOV (1986) showed in a study performed in a slaughterhouse in Lyon that with traditional meat inspection methods a prevalence of 2% for bovine cysticercosis was calculated, whereas using additional incisions in different parts of the carcass increased this rate up to 22.0%. Almost the same results were reported by MINOZZO et al. (2002). The sites where the cysts have been detected were just the locations at the carcass that are examined routinely at the slaughterhouse. It is considered that if a cyst is found, this indicates infestation and more cysts are proba-bly present (WILSON 2005).

Size of the cysts: in general, the constituents of larger cysts can easier be examined visually. The low percentage of small cysts (15.6%) within this study might be due to the fact that small cysts are not easily detected during the visual examination of the carcass, so that they escape detection. Furthermore, small cysts might be misdiag-nosed with other alterations as small abscesses or other parasites as Sarcocystis as an example.

Constituents of the cysts: as it was shown with the morphological examination of the cysts collected at the participating slaughterhouses in this study, many degenera-tive cysts, either cheesy or calcified, and also dull cysts, were visually diagnosed as T. saginata metacestodes, although they have no further features confirming that they belong to this parasite. Furthermore some infested animals had two cysts with different morphological appearance.

The percentages of the viable cysts and the degenerative cysts detected in the slaughterhouse of Berlin/Spandau were 29.6 and 70.4%, from 1981 to 1988 respec-tively (GOETZE 1989). Comparing these results with the percentages of viable cysts and degenerative cysts detected during the present study (7.3 and 81.3%

respec-tively) indicates that viable cysts have been detected about four times more frequent than the viable cysts from the cooperated slaughterhouses with this thesis. This can be attributed to a fresh infestation or even a kind of repetitive infestation, as many of these animals had viable cysts and degenerative cysts at the same time. Further, the reported prevalence of bovine cysticercosis in the slaughterhouse of Berlin was 4.8%

for the mentioned period for both light and intensive infestations (GOETZE 1989).

This is a significant higher percentage compared with the 0.5% and 1.1% found in this study. Although there should be a solid immunity after the first infestation, but the possibility of re-infection should also be taken into consideration.

Gender and age of the infested animals: focussing on the gender and the age of the animals harbouring cysts in this study there seemed to be a correlation between gender/ age and the level of infestation with T. saginata cysticercosis.

Statistical data from the federal states Bremen and Mecklenburg-Vorpommern indi-cated that in 2005, 37.3% of the slaughtered animals were males, whereas 62.7%

were females (STATISTISCHES BUNDESAMT 2006). Comparing these data with the number of the male and female animals that harboured cysts in this study, the relative percentage of infested male animals was quite high. These results reflect partially the results obtained from earlier studies (OSTERTAG 1902, OSTERTAG 1932, WHO 1957), where most of the cysts had been detected in young male ani-mals.

This observation seems to be in contrast with the fact, that the risk of infection through grazing is smaller in male animals than for females. The main route of infec-tion of the grazing cows is the contaminated pastureland that is polluted by sewage or flood water (WHO 1957). On the other hand, the main route of infection of young male animals were thought to be the farmers hands, contaminated with the eggs of the parasite, failing to remove all traces of their faeces before feeding calves (WHO 1957).

Furthermore it was noticeable, that a high percentage of males younger than 25 months harboured viable cysts, in contrast to the females. This can be explained by

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took place, and the immunity of the young animal body did not have time to react enough against the cysts either through caseation, or calcification. In general, milk cows are reared for longer time than the male animals, making them more suscepti-ble to infection and the build up of immunity against this metacestode. Furthermore, although there is no evidence that the immunity of male bovines reacts in a different manner against cysticercosis from the females, this possibility should be taken into consideration and should also be studied.

It can also be concluded from the morphological examination of the cysts that it should be no longer dependent on the static description of the outer shapes and in-ternal anatomy of the cysts to claim their belonging to T. saginata cysticercosis or not. Other methods should be used to confirm or reject this slaughterhouse diagno-sis. As it was shown from the morphological results of this study, detailed morpho-logical examination of the cysts alone could not confirm or reject the meat inspector’s diagnosis.

7.2 PCR

Sensitivity and specificity of the primers: the used PCR could detect small amounts of T. saginata DNA that reaches to 200fg. In comparison with 8pg-DNA found in one T. saginata egg (GONZALEZ et al. 2002) or with the average amount of extractable DNA from positive field cysts (5.83 µg), this indicates a high sensitivity level of the used primers.

Results obtained from the use of other primers (VON NICKISCH ROSENEGK et al.

1999, MAYTA et al. 2000, YAMASAKI et al. 2004) were either not species-specific or need further evaluation.

The fact that there were viable cysts with negative PCR results with both primers could be attributed to the hypothesis that these can be cysts or lesions of other para-sites, which can be found in muscles. The examination of cysts collected in New Zea-land and SwitzerZea-land between 1995 and 1996 with PCR, showed that cysticerci which appeared not to be caused by T. saginata, can be found in cattle, although they resemble T. saginata metacestodes both in gross pathology and histology (VAN

DER LOGT and GOTTSTEIN 2000). This alerted regulatory authorities to possible existence other cestodes or helminthes.

Furthermore, although both primers were tested for specificity and sensitivity (GON-ZALES et al. 2000, 2002), the appearance of these negative samples raises the pos-sibility of false negative results using both primer sets, presence of inhibitors that in-terfere with PCR amplification, or it may be attributed to insufficient amplifiable DNA in positive, but degenerate specimens. These equivocal points need to be clarified through further evaluation of the used primers in terms of defined positive and nega-tive samples.

Confirmation of the morphological results: the PCR results did not mainly depend on the morphological results, as many dull or calcified cysts, that seemed to have no connection with T. saginata cysts, showed positive results with PCR and vice versa.

This emphasized that these deformed cysts really belong to T. saginata metacesto-des, or they might be false positive samples that were misdiagnosed as positive with the PCR.

It was shown during this study, that although PCR is an expensive and time consum-ing test, it is an applicable and sensitive method for the forensic confirmation of the slaughterhouse diagnosis, and to exclude false positive diagnosed cysts using the traditional meat inspection methods (see attachment 2).